The main cause of death in patients with cancer is the invasion of body tissues by metastasizing tumor cells. Migration and invasion by tumor cells during metastasis require the coordination of cytoskeletal networks to form polarized structures, such as lamellipodia and invadopodia. Vimentin expression is a hallmark of the epithelial-mesenchymal transition in carcinoma cells and correlates strongly with invasive potential. Although recent studies demonstrate that vimentin regulates lamellipodia formation and invadopodia elongation, the mechanisms through which vimentin exerts influence over actin assembly are not well understood; however, CARMIL2 is a strong candidate to play a key role. CARMIL2 localizes to vimentin filaments in cells and regulates capping protein (CP), a key regulator of actin networks, in actin polymerization assays. CARMIL2 knockdown cells exhibit defective migration, diminished lamellipodial ruffling and macropinocytosis, and possess a striking multi-lamellipodia phenotype. How vimentin localization relates to CARMIL2 function and the importance of CP regulation by CARMIL2 for cell migration and invasion are not established. Additionally, CARMIL2 possesses a CARMIL homology domain (CHD) of unknown function. The high level of sequence similarity of the CHD among CARMIL family members suggests that it serves a conserved role. The central hypothesis of this proposal is that CARMIL2 regulates the polarity and dynamics of the actin network during cell migration and invasion by regulating CP and coordinating actin filament dynamics with the vimentin network. The following specific aims will test this hypothesis using molecular, cell biological, and biochemical approaches: 1) To test the importance of CP regulation by CARMIL2 for cell migration and invasion; 2) To test the importance of CARMIL2 localization to vimentin filaments for cell migration and invasion; 3) To identify proteins that interact with the conserved CHD of CARMIL2. The long-term objective of this project is to elucidate how cells assemble polarized cytoskeletal networks to drive cell migration and invasion during tumor metastasis. Completion of this proposal will provide an improved understanding of these mechanisms and will aid in the search for new cancer therapies. PUBLIC HEALTH RELEVANCE: The main cause of death in patients with cancer is the invasion of body tissues by metastasizing tumor cells. This proposal explores the molecular basis by which cells polarize and assemble cytoskeletal networks to drive migration and invasion during tumor metastasis. Completion of this proposal will provide an improved understanding of metastasis and aid in the search for new cancer therapies.